The overall, long-term objective of this project is elucidation of the biochemical mechanisms underlying granulocyte proliferation and differentiation. Our approach is to define, at the molecular level, the regulatory role of colony stimulating activity (CSA), the presumptive primary inducer of normal granulopoietic and leukemic cell differentiation. Implementation of this approach centers around development of convenient and quantitative radioimmunoassays (RIA) and immunocytofluorescence (IF) procedures for studying the CSA-dependent synthesis of granulocyte-specific protein in a readily accessible animal model, the mouse. In particular, we are studying the proteins myeloperoxidase and lactoferrin. Each is synthesized at a different period of the granulocyte maturation sequence, and consequently, each is initially associated with a different recognizable cell type (promyelocyte and myelocyte, respectively). We have demonstrated a linear dose-response relationship at both the biochemical (RIA) and cellular (IF) levels between the net synthesis of LF and the concentration of CSA (endotoxin-treated mouse lung conditioned medium) in liquid cultures of murine bone marrow cells (1 ml, slide chamber,less than or equal to 1.25 x 10 to the 4th power target cells/ml). The magnitude of the dose-response (RIA) between day 3 and day 5 increased approximately 3-fold as CSA concentration increased from 4% to 8% (v/v). IF studies using a fluorescein-conjugated F(ab')2 fragment of rabbit anti-LF IgG demonstrated that the number of LF-containing cells and the total number of cells per culture increase (each about 2.5-fold) as CSA went from 0.4% to 6.4%. The latter response (total cells) was more pronounced because the marrow cell population contained precursor cells which proliferated in response to CSA, but whose progeny did not contain LF (neutrophils less mature than myelocytes, and all cells of the monocyte/ macrophage series). In early cultures (less than or equal to 72 hr) we observed that within individual clones of neutrophils, the proportion of cells which contained LF varied from 0 to 1, with some clones consisting of both LF positive and LF negative cells. This latter phenomenon indicated a pattern of asynchronous differentiation. We have purified murine neutrophilic MPO to an RZ value of 0.83, and h (Text Truncated - Exceeds Capacity)